mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Genomic analysis of P. aeruginosa from chronically infected CF patients
Background Pseudomonas aeruginosa ''is an opportunistic pathogen capable of causing acute and chronic infections in a variety of host organisims, ranging from plants to humans1. In humans, ''P. aeruginosa ''is typically associated with establishing chronic lung infections in individuals suffering from Cystic Fibrosis, and once established, these infections directly correlate with the morbidity and mortality associated with progression of the disease 2. Since these infections typically last the duration of a Cystic Fibrosis patients life, they offer a unique opportunity to study how opportunistic bacterial pathogens accumulate mutations overtime and in doing so, can provide crtical insight into which genetic modifications facilitate adaptation and virulance within a human host. Overview In a recent study by Marvig RL et. al, the authors performed a genome wide sequencing study using 55 ''P. aeruginosa ''DK2 isolates collected from 21 individuals in Denmark over a 38 year period. Through phlylogenetic analysis, they were able to detect 8,530 mutations within the genomes of the isolates they examined. In doing so the authors were able to trace the ancestral DK2 clone's spread among different Cystic Fibrosis patients through several transmission events, and then track the resulting rise of unique hypermutating lineages within some individuals through parallel evolution. Interestingly, the authors observed that in many of the hypermutating strains, two "pathoadaptive mutations" accumulated within genes encoding antibiotic resistance, cell envelope proteins, and genes encoding transcription factors. The methods utilized and the results from this study are described below in greater detail. Methods '''Strains & genome sequencing' The 55 strains the authors used in this study were collected over a 38 year period from the sputum of 21 patients with Cystic Fibrosis. The genomic DNA was isolated from each sample and sequenced using an illumina based platform, producing 100 bp reads with an average coverage depth between 63 and 213 fold. 'Detection of mutations ' The reads from each sample were then mapped against the P. aeruginosa ''DK2 reference genome, and the single nucleotide polymorphisms (SNPs) were identified using the varFilter algorithm in SAMtools. 'Evolutionary analysis' The evolutionary rates and diversion times of each strain were determined through Bayesian analysis with the BEAST software package and the isolate: CF510-2006 as an outrgroup. Phylogenetic trees were created off of this data using the TreeAnnotator program of the BEAST software package. Major results The authors identified 8,530 mutations within the 55 isolates they sequenced, of which 7,326 mutations were found to be unique SNPs. When the data was compiled into a phylogenetic tree, the authors were able to observe a linear relationship between the time of sampling and the number of mutations accumulated compared to the ancestral DK2 strain in 44 of the isolates. In addition, the data suggested that The average mutation rate in these isolates was estimated to be 2.6 SNPs/year which is similar to that observed in other pathogenic bacteria such as ''Shigella sonnei ''and ''Vibrio cholerae 2,6. Interestingly, 11 of the isolates collected at later timepoints from 10 patients were found to have accumulated mutations at an excessively high rate, with total SNP's ranging from 284 to 2,564 per isolate. The authors termed these strains, "hypermutators" and through further analysis, found that the majority had aquired mutations in the mismatch repair (MMR) or error repair machinery. More specifically, one isolate had a nonsynonomous mutation in mutY, two of the isolates had non synonomous mutations within mutS, ''four isolates had non-synonomous mutations in ''mutL, ''while one had mutations in both mutS & mutL.'' It was noted that within the hypermutator strains, mutations were skewed towards regions of the genome containing strings of the same nucleotide (refered to as homopolymers), which are more prevelant in genes typically expressed on the envelope surface, or within antibiotic resistance genes. Interestingly, the authors observed that 65 genes were mutated in two or more of the hypermutator isolates, including the 14 antibiotic resistance genes: ''ampC, emrB, ftsI, fusA, gyrA/B, mexB/Y, pmrB, pprA, oprD, and rpoB/C, ''along with four uncharacterized transcriptional regulators and numerous outter membrane proteins. Additonally, all of the hypermutator strains were found to have nonsynonomous mutations within two uncharacterized genes predicted to be expressed as receptors on the out cell membrane. As a whole this data suggests that genes containing homopolynucleotide tracts might act as mutation "hotspots" to allow bacteria to rapidly adapt to their host environment 4. References 1 http://en.wikipedia.org/wiki/Pseudomonas_aeruginosa 2 Yang L, Jelsbak L, Marvig RL, Damkiaer S, Workman CT, et al. (2011) Evolutionary dynamics of bacteria in a human host environment. Proc Natl Acad Sci U S A 108: 7481–7486. 3 Marvig, R. L. et al. Genome analysis of a transmissible lineage of Pseudomonas aeruginosa reveals pathoadaptive mutations and distinct evolutionary paths of hypermutators. PLOS Genet. 9, e1003741 (2013). 4 Moxon ER, Rainey PB, Nowak MA, Lenski RE (1994) Adaptive evolution of highly mutable loci in pathogenic bacteria. Curr Biol 4: 24–33.